Lignocellulose is the main component of forest product residues and agricultural waste. Lignocellulosic raw materials are mainly composed of cellulose, hemicellulose, and lignin. The cellulose fraction is made up of glucose polymers, whereas the hemicellulose fraction is made up of a mixture of glucose, galactose, mannose, xylose and arabinose polymers. The lignin fraction is a polymer of phenolic compounds. Xylose is found in hardwood and softwood hemicelluloses, whereas arabinose is a component in hemicellulose in certain agricultural crops, such as corn.
The cellulose and hemicellulose fractions can be hydrolyzed to monomeric sugars, which can be fermented to ethanol. Ethanol can serve as an environmentally acceptable liquid fuel for transportation, since carbon dioxide released in the fermentation and combustion processes will be sorbed by growing plants in forests and fields.
The price for lignocellulose-derived ethanol has been estimated by Von Sivers, M., G. Zacchi, L. Olsson, and B. Hahn-Hägerdal. 1994. ‘Cost analysis of ethanol production from willow using recombinant Escherichia coli.’ Biotechnol. Prog. 10:555-560. Their calculations were based on the fermentation of all hexose sugars (glucose, galactose and mannose) to ethanol and they estimated that the fermentation of pentose sugars (xylose and arabinose) to ethanol could reduce the price of ethanol by approximately 25%. The microbial conversion of lignocellulosic derived hexoses and pentoses would therefore not only be environmentally acceptable, but could also be cost-effective.
Further, the release of monomeric sugars from lignocellulosic raw materials also release by-products, such as weak acids, furans and phenolic compounds, which are inhibitory to the fermentation process. The commonly used Baker's yeast, Saccharomyces cerevisiae, is the only ethanol producing microorganism that is capable of efficiently fermenting non-detoxified ligocellulose hydrolysates (Oisson and Hahn-Hägerdal, “Fermentation of lignocellulosic hydrolysates for ethanol production”, Enzyme Microbial Technol. 18;312-331, 1996). Particularly, efficient fermenting strains of Saccharomyces cerevisiae have been isolated from the fermentation plant at a pulp and paper mill (Lindén et al., “Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant”, Appl. Environ. Microbiol. 158:1661-1669, 1992).
Saccharomyces cerevisiae ferments the hexose sugars glucose, galactose and mannose to provide ethanol, but is unable to ferment the pentose sugars xylose and arabinose due to the lack of one or more enzymatic steps. Saccharomyces cerevisiae can ferment xylulose, an isomerization product of xylose, to ethanol (Wang et al., “Fermentation of a pentose by yeasts”, Biochem. Biophys. Res. Commun. 94:248-254, 1980; Chiang et al., “D-Xylulose fermentation to ethanol by Saccharomyces cerevisiae”, Appl Environ. Microbiol. 42:284-289, 1981; Senac and Hahn-Hägerdal, “Intermediary metabolite concentrations in xylulose- and glucose-fermenting Saccharomyces cerevisiae cells”, Appl. Environ. Microbiol. 56:120-126, 1990).
In eukaryotic cells, the initial metabolism of xylose is catalyzed by a xylose reductase (XR), which reduces xylose to xylitol, and a xylitol dehydrogenase (XDH), which oxidizes xylitol to xylulose. Xylulose is phosphorylated to xylulose 5-phosphate by a xylulose kinase (XK) and further metabolized through the pentose phosphate pathway and glycolysis to ethanol.
Saccharomyces cerevisiae has been genetically engineered to metabolize and ferment xylose. The genes for XR and XDH from the xylose-fermenting yeast Pichia stipitis have been expressed in Saccharomyces cerevisiae (European Patent to C. Hollenberg, 1991; Hallborn et al., “Recombinant yeasts containing the DNA sequences coding for xylose reductase and xylitol dehydrogenase enzymes”, W091/15588; Kötter and Ciriacy, “Xylose fermentation by Saccharomyces cerevisiae”, Appl. Microbiol. Biotechnol. 38:776-783: 1993). The transformants metabolize xylose but do not ferment the pentose sugar to ethanol.
The gene for xylulose kinase (M) from Saccharomyces cerevisiae has been cloned and overexpressed in XR-XDH-expressing transformants of Saccharomyces cerevisiae (Deng and Ho, “Xylulokinase activity in various yeasts including Saccharomyces cerevisiae containing the cloned xylulokinase gene”, Appl. Biochem. Biotechnol. 24/25:193-199, 1990; Ho and Tsao, “Recombinant yeasts for effective fermentation of glucose and xylose”, W095/13362, 1995; Moniruzzarnan et al., “Fermentation of corn fibre sugars by an engineered xylose utilizing a Saccharomyces strain”, World J. Microbiol. Biotechnol. 13:341-346, 1997). These strains have been shown to produce net quantities of ethanol in fermentations of mixtures of xylose and glucose. Using the well established ribosomal integration protocol, the genes have been chromosomally integrated to generate strains that can be used in complex media without selection pressure (Ho and Chen, “Stable recombinant yeasts for fermenting xylose to ethanol”, W09/42307; Toon et al., “Enhanced cofermentation of glucose and xylose by recombinant Saccharomyces yeast strains in batch and continuous operating modes”, Appl. Biochem. Biotechnol. 63/65:243-255,1997).
Although great strides hare been made, there exists a need in the art for a method of, and a tool for efficiently fermenting lignocellulose hydrolysates to produce ethanol.